Aeromonas sobria as a potential candidate for bioremediation of heavy metal from contaminated environments

The uncontrolled discharge of industrial wastes causes the accumulation of high heavy metal concentrations in soil and water, leading to many health issues. In the present study, a Gram-negative Aeromonas sobria was isolated from heavily contaminated soil in the Tanjaro area, southwest of Sulaymaniyah city in the Kurdistan Region of Iraq; then, we assessed its ability to uptake heavy metals. A. sobria was molecularly identified based on the partial amplification of 16S rRNA using novel primers. The sequence was aligned with 33 strains to analyze phylogenetic relationships by maximum likelihood. Based on maximum tolerance concentration (MTC), A. sobria could withstand Zn, Cu, and Ni at concentrations of 5, 6, and 8 mM, respectively. ICP-OES data confirmed that A. sobria reduced 54.89% (0.549 mM) of the Cu, 62.33% (0.623 mM) of the Ni, and 36.41% (0.364 mM) of the Zn after 72 h in the culture medium. Transmission electron microscopy (TEM) showed that A. sobria accumulated both Cu and Ni, whereas biosorption was suggested for the Zn. These findings suggest that metal-resistant A. sobria could be a promising candidate for heavy metal bioremediation in polluted areas. However, more broadly, research is required to assess the feasibility of exploiting A. sobria in situ.

www.nature.com/scientificreports/ Excavation and solidification/stabilization are the traditional approaches for remediating heavy metals-contaminated sites. These techniques effectively control contamination but do not entirely remove heavy metals 12 . However, they, have significant drawbacks, such as limitations on cost-effectiveness, the production of hazardous byproducts, or inefficiency 13 . Therefore, various other technologies have been applied to reduce heavy metal toxicity from contaminated environments [14][15][16][17] . Physicochemical methods such as precipitation, electro-winning, ion exchange, soil replacement, electrocoagulation, membrane filtration, electrodialysis, and activated carbon are mainly used to reduce heavy metals from polluted wastewater 18 . Instead, researchers have focused on bioremediation, an environmentally-friendly and cost-effective approach, to remove heavy metals; biological systems such as plants, algae, fungi, and bacteria are effectively used to remove, degrade or immobilize toxic pollutants from the contaminated environment 15,19 . In contrast to conventional physicochemical methods, using microbial metabolic abilities to degrade or remove environmental toxins offers a cost-effective and safe solution. Although present in the environment, very diverse and specialized microbial consortia effectively eliminate many contaminants 20 . Nevertheless, biological techniques are simple and do not produce in secondary pollution 13 .
Aeromonas belongs to the class gamma proteobacteria within the Aeromonadaceae family 21 . Aeromonas is Gram-negative, rod-shaped, non-spore-forming, and facultatively anaerobe. Aeromonas species are common in soil and aquatic habitats, on a variety of foods, as well as in invertebrate and vertebrate animals 22 . A few strains of Aeromonas cause infections in poikilothermic animals, including mammals, birds, and fish. Human infections by these strains include septicemia, pneumonia, wound infections, urinary tract infections, and gastroenteritis 23 . In addition, Aeromonas species have an extraordinarily efficient ability to transform and remove heavy metals and other contaminants from polluted areas 24,25 . For example, species of Aeromonas can degrade oil hydrocarbons 26 , decolorize triphenylmethane dyes such as malachite green 27 , and remove nitrates from wastewater 28 .
It is beyond the scope of this study to investigate the accumulation of heavy metals in the vegetation and soil surface of the Tanjaro area, particularly the analysis of Zn, Cu, and Ni; however, due to the various sources of such metals, this could be problematic. Heavy metals may be emitted from a variety of sources in the Tanjaro area, including local and public generators, as well as the presence of industrial factories. Further data collection is required to determine exactly how these metals accumulate and affect the environment as a whole. Notwithstanding these limitations, the study suggests that the metals in the plantations need to be evaluated. Then, in a more comprehensive assessment, A. sobria could play a role in removing these metals, allowing researchers to fabricate and produce abundant and environmentally viable sources of A. sobria monoculture system for remediating heavy metal-contaminated areas. With that in mind, the present study aims to isolate indigenous heavy metal-tolerant A. sobria from the soil and to investigate its ability to minimize the toxicity of Cu, Ni, and Zn.

Results
Isolation of metal-resistant A. sobria. A. sobria was isolated from soil contaminated with heavy metals, identified biochemically (Supplementary Table S1) and confirmed molecularly. Phylogenetic analysis based on the 16S rRNA gene sequence was performed. A partial sequence of 16S rRNA ( Supplementary Fig. S1) of isolate KQ_21 was assigned to the species A. sobria, which belongs to the Proteobacteria, subgroup Gamma, and the family Aeromonadaceae (Fig. 1). The result reveals that the KQ_21 isolate is the closest to A. sobria strain 208.

Evaluation of heavy metal removal by A. sobria.
A. sobria tolerated high Cu concentrations and had an average growth from 1 to 4 mM. However, the Cu metal showed minimum inhibitory concentration (MIC) at 5 mM and MTC at 6 mM ( Fig. 2). For Ni, A. sobria tolerated up to 8 mM and showed a MIC of 6 mM. Zn showed higher toxicity to A. sobria than the previously mentioned two heavy metals. A. sobria was able to grow at 5 mM of Zn; however, the MIC appeared at 3 mM and completely inhibited the growth at 6 mM (

Mechanism of heavy metal uptake by A. sobria. A. sobria is substantially tolerant to relatively high
concentrations of heavy metal. The Cu and Ni metals were reduced and accumulated inside the cells (Fig. 4b,c) in comparison to A. sobria free from metals (Fig. 4a). Furthermore, biosorption is the most probable mechanism for Zn ( Fig. 4d).

Discussion
The Tanjaro River is permanent river in Sulaymaniyah city; it is an essential source of industry and agriculture. The river is approximately 58 km long and is located southwest of Sulaymaniyah by 7 km. It arises through the confluence of the Qilyasan and Kani-Ban small rivers. Also, other small tributaries and springs discharge into the river. It flows north to south, passing through three small towns (Kani Goma, Bakrajo, and Zarayan) before emptying into the Darbandikhan Dam reservoir. Around 900 factories are located near the Tanjaro River. The heavy metal-infested waste caused by these factories is hazardous. Therefore, utilizing microorganisms capable of mitigating heavy metals is an efficient way of cleaning up the contaminated sites from the toxicity of such pollutants 29 . Some microorganisms can thrive in metal-polluted areas using a variety of mechanisms. Metals transformation, e.g. formation of metal oxalate complexes, is one of the common mechanisms used to avoid the toxicity of metals by the action of microbes 2 . Tanjaro is a heavily contaminated area in Sulaymaniyah city. It has been reported that numerous anthropogenic activities such as agriculture, aquaculture, and hospital run-off spill www.nature.com/scientificreports/ directly into the water, causing heavy metal buildup inside the river. The polluted water is subsequently utilized for farm, animal, and irrigation purposes, leading to harmful effects on human and animal health 5 .
To the best of our knowledge, no report mentions Aeromonas sobria as a new candidate for heavy metals removal. Aeromonas species generally associated with human infections include A. caviae, A. schubertii, A. hydrophila, A. veronii (biovars veronii and sobria), and A. jandaei 30 ; however, biochemically distinct A. veronii biovar sobria have been predominantly found in clinical isolates 31 . Our results show that A. sobria can tolerate high concentrations of different heavy metals. The genus Aeromonas has more than 33 recognized species 32 . Most Aeromonas species are able to grow in diverse environments such as soil and sediments 33 . In the present study, 16S rRNA sequence analysis results indicate that A. sobria KQ 21 belongs to the same evolutionary branch as the wastewater strain A. sobria 208. Phylogenetic analysis showed the separation of species from the genus Aeromonas into different distinguished clades, suggesting that a diverse array of species might emerge or separate as a distinct related genus in future studies.
Aeromonas from the Tanjaro region tolerated high concentrations of Cu (6 mM), Ni (8 mM), and Zn (5 mM). Ni is tolerated at higher concentrations by A. sobria, possibly due to its low atomic weight, strong electronegativity, and small ionic radius. These ions well-suited to being trapped by bacterial biomass 34 . Previous studies proved that Aeromonas potentially resists different concentrations of metals such as Fe, Zn, Pb, Cd, Ni, and As 35 . The current study results show that reduced Cu, Ni, and Zn gradually increased with increasing incubation periods; www.nature.com/scientificreports/ the rate of reduced heavy metals was the highest in the first 24 h of incubation. The loss of viability observed after a few days in the stationary phase could result from random cellular damage or an altruistic death response to feeding the few survivors 36 . Furthermore, oxidative damage causes cellular deterioration in the stationary phase, decreasing the bacterial population, which ultimately influences the heavy metal reduction process 37 . The ability of Aeromonas strains to persist in the presence of heavy metals may be due to the possession of plasmids carrying heavy metal-resistant genes 38 . Aeromonas uses different mechanisms to reduce heavy metal toxicity, including biosorption. Unspecific heavy metal ions bind to extracellular and cell surface-associated polysaccharides and other proteins as part of a non-enzymatic mechanism. These molecules contribute to the persistence of Aeromonas in heavy metal-contaminated environments 35 . The trapping of metals by negatively charged groups such as phosphoryl, hydroxyl, and carboxyl on the cell wall of Aeromonas is a widely used mechanism of resistance to heavy metals 39 . Numerous factors affect the biosorption mechanism comprising temperature, particle size, ionic strength, and biomass concentration 40 . Our isolate tolerates a higher concentration of heavy metals than Aeromonas isolates reported by previous studies 39, 41 . Several scholars recommended Aeromonas  www.nature.com/scientificreports/ as a potential system for the remediation of different chemicals, including triarylmethane dyes and polycyclic aromatic hydrocarbons 24,25,42,43 .
In the current study, TEM analysis of A. sobria KQ_21 showed intracellular and periplasmic accumulation of Cu, Ni, and Zn. Similarly, heavy metal transport through bioaccumulation has been reported in a variety of bacteria, including Pseudomonas aeruginosa (uranium), Citrobacter sp. (lead and cadmium), Micrococcus luteus (strontium), and Pseudomonas putida [44][45][46] . A. sobria KQ_21 shows invaluable potential to remove heavy metal toxicity compared to other Aeromonas strains reported by a previous study 39 . The ability of gram-negative bacteria to accumulate metal is often greater than that of gram-positive isolates, which could be related to differences in cell wall composition 41 . Bacterial cell walls, which are primarily composed of polysaccharides, proteins, and lipids, harbor several functional groups such as hydroxyl, carboxylate, phosphate, and amino groups that are capable of binding heavy metal ions 47 .

Materials and methods
Isolation and identification of A. sobria. To reduce heavy metal sensitive isolates, soil samples from the Tanjaro river, southwest of Sulaymaniyah city in the Kurdistan Region of Iraq (Fig. 5)  Biochemical tests were performed for the isolated dominant colonies 48 . The isolated bacteria were further confirmed with the VITEK 2 instrument (bioMérieux, USA) using a Gram-negative VITEK 2 ID card. Moreover, the selected A. sobria isolate was subjected to molecular identification by partial amplification and sequencing of 16S rRNA. Aeromonas hydrophila and Escherichia coli were used as positive and negative controls, respectively. 16S rRNA sequences of 33 Aeromonas species were downloaded from NCBI and aligned using Clustal Omega. A primer set was then designed; Aero_F (5′ TAC GGG AGG CAG CAGTG 3′) and Aero_R (5′ CAC ATG CTC CAC CGC TTG 3′) with a PCR product size of ~ 600 bp. The genomic DNA was extracted using the QIAamp Mini kit www.nature.com/scientificreports/ (Qiagen, Hilden, Germany) and amplified using PCR. The PCR amplification protocol includes initial denaturation at 94 °C for 5 min followed by 25 cycles of three stages (denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, and extension at 72 °C for 1 min). This was followed by a final extension at 72 °C for 5 min. Finally, the PCR product was run on 1% gel agarose at 80 V for 50 min. The image of the gel was captured using MultiDoc-It Imaging System (UVP, USA). The PCR product was sequenced (Macrogen, South Korea) and aligned with the 16S rRNA sequences of all species of Aeromonas. Multiple sequence alignment was performed using MEGA version X 49 . The phylogenetic tree was constructed using the neighbor-joining method. The unaligned regions were excluded from the analysis. E. coli was used as an outgroup.

Mechanism of heavy metal reduction.
To determine the exact mechanism of heavy metal removal, the NB was inoculated with A. sobria. The NB was then incubated at 30 °C until bacterial growth reached 0.6 OD at 600 nm. Afterward, 1 mM of Cu, Ni, and Zn was added to the medium and incubated overnight at 30 °C (modified procedure of 52 ). A glass vial containing 3% glutaraldehyde was filled with the cell suspension and left for two hours at room temperature before incubating at 4 °C overnight. The vial was washed with a cacodylate buffer before another fixation step with 2% osmium tetroxide for two hours. The glass vial was rinsed yet again with the buffer mentioned above. For infiltrating epoxy resin, fixed samples were ethanol-dehydrated. Resin-embedded  www.nature.com/scientificreports/ samples were ultimately polymerized with mild heat. The samples were post-stained with uranyl acetate followed by lead citrate before being examined with TEM. (JEOL, Japan) (Modified procedure of 53 ).

Conclusions
The surrounding Tanjaro area is known as the industrialized district due to many factories; these premises are abundant mineral resources. Metal leaches cause severe heavy metal pollution to the Tanjaro River and aquatic ecosystems. Heavy metal-contaminated areas harbor microbial resistance toward toxic metals. For the first time, in the framework of this study, evaluation of the heavy metal removal by the action of A. sobria is determined. Isolation and molecular identification of A. sobria from highly heavy metal-polluted soil in the Tanjaro area were reported. A. sobria KQ_21 exerts maximum tolerance against Cu, Ni, and Zn. A. sobria uses different mechanisms for reducing heavy metal toxicity. TEM revealed bioaccumulation and biosorption mechanisms to remove heavy metals. ICP showed the maximum reduction of heavy metals in different time frames. This investigation advocates the use of A. sobria as a novel candidate for heavy-metal bioremediation. Further studies are required to ascertain the effectiveness of A. sobria in removing heavy metals as well as other pollutants from contaminated areas.

Data availability
All data generated or analyzed during this study are included in this published article (and its supplementary information files).